Lyme disease is the most common tick-borne illness in the United States and Europe. It is caused by Borrelia burgdorferi, a bacterial pathogen that is maintained in nature in a zoonotic cycle between various species of small mammals and an ixodid tick vector. A hallmark of the Lyme disease spirochete is its unusual segmented genome, which includes a large number of linear and circular plasmids. Increasing evidence indicates that plasmid-encoded functions are critical for successful adaptation to the different environments that B. burgdorferi encounters during its infectious cycle. We have developed genetic tools to investigate basic aspects of the unusual genomic organization, cellular structure and metabolism of B. burgdorferi. We have extended this investigation to an in vivo setting with an experimental system that closely mimics the natural arthropod vector/rodent host infectious cycle. Through an understanding of the basic molecular biology of the organism, we hope to gain insight into the infectious strategy utilized by this significant vector-borne pathogen and thereby facilitate efforts to prevent, diagnose and treat Lyme disease. Genetic manipulation of B. burgdorferi is currently extremely inefficient, requiring microgram quantities of DNA, yet yielding only a few transformants. This prevents the routine generation of saturated mutant libraries or introduction of complete genomic libraries in B. burgdorferi, thus severely limiting the application of effective genetic screens to the Lyme disease spirochete. Endogenous plasmid-encoded restriction/modification (R/M) systems constitute part of the barrier to stable introduction of foreign DNA in B. burgdorferi. In FY2017 we collaborated with Dr. Fang Gang at Icahn School of Medicine at Mt. Sinai to identify the DNA sequence motifs recognized by these R/M systems of B. burgdorferi. Armed with this information, we are currently designing shuttle vectors and selectable markers that lack these recently identified R/M sites of B. burgdorferi. Future studies will assess the utility of these sequence-optimized constructs for highly efficient transformation of B. burgdorferi and their potential to greatly expand genetic studies in the Lyme disease spirochete. Transmission of B. burgdorferi in nature requires ingestion of vertebrate blood by ticks, which not only acts as a means of spirochete transfer between the tick vector and vertebrate host, but was also shown in early studies to stimulate and support spirochete replication within feeding ticks. Subsequent studies have demonstrated that in addition to stimulating replication, the encounter with host blood by resident B. burgdorferi in the midgut of feeding ticks initiates a critical adaptive response that prepares the spirochete for vertebrate infection, including induction of the virulence determinant OspC. A recent study from our lab, published in 2016, demonstrated that B. burgdorferi present in unfed ticks are essentially non-infectious, even when the contents of multiple ticks were pooled to provide a large inoculum of 10,000 viable bacteria. In contrast, mouse infection was consistently established with a much smaller inoculum (30 spirochetes) derived from ticks after they had fed. We concluded that in addition to stimulating spirochete replication, exposure to vertebrate blood during tick feeding also induces phenotypic changes that conditionally prime B. burgdorferi for subsequent infection of a vertebrate host. In FY2017, we have extended our previous observation of conditional priming of B. burgdorferi to an investigation of how host immune status impacts the virulence of spirochetes within infected ticks. In nature, multiple strains of B. burgdorferi are stably maintained at high prevalence in both the tick vector and reservoir hosts sharing the same local geographic area. In an endemic region, infected ticks would be expected to feed on infected hosts carrying the same or different B. burgdorferi strains. We show that ingestion of blood from an infected host can severely restrict the virulence of spirochetes residing in the tick midgut and thereby prevent super-infection of the host by the same B. burgdorferi strain. This attenuation of spirochete infectivity was not observed when infected ticks and blood-meal hosts carried different B. burgdorferi strains, or when immune-deficient SCID mice replaced wild type mice in similar experiments. OspC is a known target of strain-specific neutralizing antibodies elicited during infection with B. burgdorferi. In FY2017, we have initiated a study to assess whether exchanging just the OspC component of the spirochete surface with that of a different strain is sufficient to allow evasion of this neutralizing immune response and transmission to an infected host. The results of our FY2017 study indicate that blood from an infected, immune host can have either a profoundly negative or positive impact on the virulence of the Lyme disease spirochete in feeding ticks. This dichotomous response to host blood prevents super-infection by the same B. burgdorferi strain, while promoting infection by heterologous strains. Significantly, this study demonstrates for the first time that protective immunity against the Lyme disease spirochete, like induction of critical virulence determinants, is mediated by host blood in the midgut of feeding ticks, prior to transmission. Our current working model posits OspC as the critical target of strain-specific host antibodies that effectively neutralize B. burgdorferi in the tick midgut. An extension of this model is the potential for spirochetes to evade host immunity through horizontal gene transfer and acquisition of a new ospC gene/serotype, for which there is abundant phylo-genomic evidence. The tick midgut is the likely site of DNA exchange between spirochetes during the natural infectious cycle because it can be colonized by relatively large numbers of spirochetes from multiple strains, co-existing in a biofilm-like matrix for many months. Future research will attempt to capture empirical evidence for the generation of virulent ospC escape mutants through horizontal gene transfer in the tick midgut.